Metal ions often play critical roles in protein structure and function. Engineered metal-binding sites in peptides and proteins have been widely used to enhance structural integrity, stabilize biologically active conformations, and confer novel enzymatic activities (Iverson, B. L., 1990; Regan, L., 1993; Kellis, J. T. Jr., 1991). Biochemical and structural analyses of transition metal coordination by proteins and peptides have traditionally focused on zinc, copper, manganese, and iron due to their roles in important biological processes (Klemba, M., 1995; Kruck, T. P. A., 1976; Lau, S., 1989; Franco, R., 1995). Other transition metals, not found in natural proteins, have coordination, isotopic, and chemical properties which make them attractive for peptide and protein engineering. Rhenium (Re) and Technetium (Tc) are group VIIB transition metals which share similar coordination geometries and form stable complexes with amine and amide nitrogens, carboxylate oxygens, and thiolate and thioether sulfurs, with a strong preference for thiolate sulfurs (Vanbilloen, H. P., 1995). Radioactive isotopes of Re and Tc have significant medical applications due to the nature of their associated radiation and physical half-life properties.
The synthesis and characterization of radiolabeled antibodies, peptides, and steroid hormones as in vivo tumor imaging and therapeutic agents are active areas of cancer research today. These molecules specifically target tumor cells by virtue of their high specificities for receptors and antigens present on the surfaces of these cells. In one commonly used approach, metallic radionuclides such as 186Re, 188Re, and 99mTc are appended to the tumor targeting molecule through bifunctional chelate groups which consist of a metal chelate and an activatable crosslinker (Bakker, W. H., 1991; Fritzberg, A. R., 1988; Liu, S., 1996). The resulting radiolabeled proteins, peptides, and small molecules are decorated with one or more chelating groups. The presence of bulky metal chelating groups and their associated crosslinkers may affect receptor affinity and biodistribution in vivo (Krenning, E. P., 1992; Wraight, E. P., 1992).
An alternative approach to the design of radiolabeled tumor imaging and therapeutic agents involves incorporating the metal directly into the molecule""s structure (Varnum, J. M., 1994; Varnum, J. M., 1996; Chi, D. Y., 1994). Metal centers with defined coordination geometries can provide a foundation for the construction of stable molecular structures which have high affinities for specific receptors or antigenic sites. Protein, peptide, and small molecule structures can be designed to use metal coordination to reduce conformational freedom, stabilize active conformations, or mimic native conformations.
Alpha melanocyte stimulating hormone (xcex1-MSH) is a tridecapeptide [Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2] that regulates skin pigmentation in most vertebrates (Hruby, V. J., 1993). The core xcex1-MSH sequence His-Phe-Arg-Trp, conserved in a number of species, has been found to be sufficient for receptor recognition (Hruby, V. J., 1993). xcex1-melanotropin is a naturally occurring tridecapeptide that specifically recognizes melanotropin receptors. Since mouse melanoma cells and human melanocytes possess a-melanotropin (xcex1-MSH) receptors, the use of radiolabeled xcex1-MSH analogs for diagnosis and treatment of xcex1-MSH positive cancers (i.e., melanoma) was hypothesized. Various synthetic a-melanotropin analogs have been prepared and characterized for a-melanotropin activity by V. J. Hruby, M. E. Hadley et al. They have reported that cyclic analogs of xcex1-MSH (as described by U.S. Pat. No. 4,485,039; 1984) display properties which increase their potency toward the xcex1-MSH receptor, prolong their activity and increase their resistance to in vivo enzymatic degradation.
In 1990, D. R. Bard, C. G. Knight and D. P. Page-Thomas filed an international patent application for targeting malignant melanoma with multiple xcex1-MSH analogs chemically attached to a chelating molecule (e.g. DTPA) which can subsequently be linked to a cytotoxic agent or radionuclide (e.g., In-111 or I-131). The literature reports by these authors describe the use of In-111-labeled analogs of the patented moieties for use as diagnostic agents for malignant melanoma.
The peptide analog used as the starting material for the Re and Tc labeling reactions was first described by Wayne L. Cody et al. as a superpotent xcex1-MSH analog with prolonged biological activity. This analog is now commercially available. However, neither the use of Re or Tc to covalently cyclize xcex1-MSH analogs, nor Re or Tc radiolabeling studies using this analog have been reported.
One radiolabeled xcex1-MSH analog has been reported as a targeting agent of the radionuclide Indium-111. This analog consists of two des-acetyl-MSH molecules crosslinked through the chelating group DPTA. In vivo work on this analog was carried out under Home Office project license number PPL 70/00499, and was reported in Bard, D. R. et al. No studies have been reported which attach medically important radionuclides to xcex1-MSH via peptide chelating groups.
The development of new diagnostic and therapeutic agents for treating cancer is an important area of research, since so many people develop cancer during their lifetimes. Melanomas are particularly difficult to treat successfully because they are very aggressive (early metastasis) and have proven resistant to general chemotherapy and external-radiation treatment. The use of specific biological molecules, such as xcex1-MSH, to target melanoma with diagnostic and radiotherapeutic agents is needed.
It would be useful to have a method of radiolabeling the peptide directly without the use of a separate chelating ligand and the peptide linkage group. This would alleviate some of the difficulties currently observed with the incorporation of these functionalities, such as lowered ED50 values and increased nonspecific localization.
According to the present invention, there is provided a compound for use as a diagnostic or therapeutic pharmaceutical consisting essentially of an alpha-melanotropin stimulating hormone analog which has integrally located a radionuclide. The radiolabeled alpha-melanotropin is administered to the body in an amount sufficient to allow uptake and retention by the tumor cells.